Biomedical Instrumentation- I

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This section covers the sources and characteristics of biomedical signals, the performance requirements and design constraints of medical instrumentation, bioelectric potentials and electrodes, physiological transducers, and the biomedical recorders ECG, EEG and EMG.

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The Generalised Medical Instrumentation System • Every medical instrument follows the same chain: measurand (the physiological quantity) → sensor/transducer → signal conditioning (amplification, filtering) → analogue-to-digital conversion → processing → display, recording, storage and transmission, with calibration, feedback and control around it and a power supply with patient isolation. • Sources of biomedical signals: bioelectric (ECG, EEG, EMG, EOG, ERG — from ionic currents across excitable membranes), bioimpedance (respiration, body composition, cardiac output), biomagnetic (MCG, MEG — extremely weak, requiring SQUID magnetometers), biomechanical (pressure, flow, displacement, force), bioacoustic (heart and breath sounds, Korotkoff sounds, Doppler), biochemical (pO₂, pCO₂, pH, glucose, electrolytes, enzymes) and biooptical (oximetry, plethysmography, fluorescence). • Measurement may be direct or indirect, invasive or non-invasive, continuous or intermittent, and in vivo or in vitro; a further distinction is between sensing, diagnostic, therapeutic and laboratory equipment.

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Signal Typical amplitude Frequency range ECG 0.5 - 5 mV (surface) 0.05 - 100 Hz (diagnostic);

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0.5-40 Hz monitoring EEG 10 - 100 μV 0.5 - 100 Hz (delta, theta, alpha, beta, gamma) EMG 0.1 - 5 mV 10 - 2,000 Hz EOG 10 - 3,500 μV DC - 100 Hz ERG 0.5 μV - 1 mV 0.2 - 200 Hz Blood pressure 0 - 400 mmHg DC - 50 Hz Respiratory rate 2 - 50 breaths/min 0.1 - 10 Hz Body temperature 32 - 40 °C DC - 0.1 Hz Performance Requirements • Static characteristics: accuracy (closeness to the true value — a measure of systematic error); precision/repeatability (closeness of repeated readings to one another — a measure of random error); resolution (the smallest detectable change); sensitivity (output per unit input, the slope of the calibration curve); linearity; hysteresis; drift (zero and sensitivity drift with time and temperature); range and span; threshold and dead zone; and reproducibility.

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An instrument may be precise but inaccurate, and precision is the prerequisite for accuracy but does not guarantee it. • Dynamic characteristics: frequency response and bandwidth, rise time, settling time, time constant, damping ratio (an underdamped system overshoots and rings; an overdamped one is sluggish;

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0.6-0.7 is the usual optimum), natural frequency and phase shift. • Statistics of measurement: a set of readings is described by its mean, median and mode, and its scatter by standard deviation and variance, with the standard error of the mean = σ/√n.

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Errors are systematic (bias, correctable by calibration) or random (scatter, reduced by averaging n readings, since random error falls as 1/√n), with gross (human) errors a third category.

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Diagnostic performance uses sensitivity (true positive rate), specificity (true negative rate), positive and negative predictive value and the ROC curve. • Noise and interference:

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50 Hz mains interference (the dominant problem, reduced by high CMRR, a driven right-leg circuit, shielded twisted leads, good electrode contact and a notch filter), motion and baseline wander, EMG contamination, electrode half-cell drift, thermal and shot noise, and electrosurgical interference.

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The figure of merit is the signal-to-noise ratio, improved by filtering, averaging and differential recording.

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Intelligent Systems and Design Constraints • Intelligent (smart) medical instrumentation incorporates a microprocessor or embedded system, giving automatic calibration and self-test, digital filtering and artefact rejection, automatic measurement and computation of derived parameters, alarms with limits, trending and storage, networking and telemetry, user interfaces and fail-safe operation, and increasingly machine-learning-based interpretation and decision support — with automated ECG interpretation the oldest example. • General constraints in the design of medical instrumentation — the standard examination list:

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(1) the measurand is usually inaccessible and measurement is often indirect;

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(2) signals are very small and buried in noise and interference;

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(3) biological variability between and within patients, so 'normal' is a range, not a value;

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(4) the measurement must not disturb the variable being measured (loading effect);

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(5) patient safety is paramount — electrical, thermal, mechanical, radiation, infection and chemical hazards;

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(6) the environment is hostile — motion, fluids, temperature and humidity, electromagnetic interference, and the need for sterilisation;

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(7) reliability, since failure may be fatal, requiring redundancy and alarms;

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(8) ease of use by clinical, not engineering, staff;

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(9) regulatory and standards compliance; and (10) cost, maintenance and calibration. • Electrical safety is the most examinable of these.

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Macroshock is current through the intact skin:

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1 mA perception, 10-20 mA 'let-go' (sustained muscle contraction), 100 mA ventricular fibrillation.

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Microshock is current delivered directly to the heart through an intracardiac catheter or pacing lead, where as little as 10 μA can cause fibrillation, which is why the CF (cardiac floating) classification exists alongside BF (body floating) and B.

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Protection rests on protective earthing, double insulation, equipotential bonding, isolated (floating) patient circuits with optical or transformer isolation, isolated power supplies with line isolation monitors, limits on leakage current, and regular electrical safety testing under IEC 60601.

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Bioelectric Potentials • As set out in Chapter 1: the resting membrane potential of about −70 to −90 mV arises from the Na⁺/K⁺ pump, the selective permeability of the membrane and impermeant intracellular anions, and is described by the Nernst equation for a single ion and the Goldman equation for the whole membrane.

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An adequate stimulus depolarises the membrane to threshold (about −55 mV), whereupon voltage-gated Na⁺ channels open (depolarisation to about +30 mV), then inactivate as K⁺ channels open (repolarisation), often with a brief hyperpolarisation. • The action potential is all-or-none, information being coded in frequency, and is followed by absolute and relative refractory periods.

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It propagates by local circuit currents — continuously in unmyelinated fibres and by saltatory conduction between nodes of Ranvier in myelinated fibres — the velocity rising with fibre diameter and myelination. • What an electrode on the skin records is not the action potential itself but the volume-conducted field produced by many cells acting together, attenuated and low-pass filtered by the intervening tissue — which is why surface signals are in the millivolt and microvolt range and why electrode placement matters so much.

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Electrodes • An electrode is a transducer that converts the ionic current of the body into the electronic current of the instrument, by means of the chemical reactions occurring at the electrode-electrolyte interface. • Half-cell potential is the DC potential developed at that interface; a difference between two electrodes produces an offset that can saturate a high-gain amplifier, and drift in it produces baseline wander.

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Electrodes are polarisable (behaving like a capacitor, no net charge crossing — platinum; large half-cell potential and motion artefact) or non-polarisable (behaving like a resistor, charge crossing freely — silver/silver chloride).

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Ag/AgCl is therefore the standard biopotential electrode, with a stable, low half-cell potential and low motion artefact.

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The equivalent circuit of an electrode is a half-cell voltage source with a parallel RC (double layer) in series with the electrolyte/skin resistance. • Skin preparation matters more than the electrode: the stratum corneum is the principal source of impedance and of motion artefact, so the skin is cleaned, lightly abraded and covered with electrolyte gel; impedance should be below about 5 kΩ for ECG and below 5-10 kΩ for EEG, and differences in impedance between electrodes are worse than high impedance itself because they degrade CMRR.

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Electrode type Description and use Plate/limb electrodes Metal plates strapped to the limbs — the original ECG electrodes Suction (bulb) electrodes For the chest leads of a resting ECG; quick to apply and move Floating (recessed) electrodes Ag/AgCl pellet recessed in a gel-filled cavity so that the metal never touches the skin — the standard disposable ECG and monitoring electrode, with minimal motion artefact Disposable pre-gelled adhesive electrodes Foam or hydrogel, single use — prevents cross-infection Dry and capacitive electrodes No gel; used in wearables and long-term monitoring, with higher impedance EEG scalp electrodes Small cup (disc) electrodes with collodion or paste, placed by the international 10-20 system; also needle and subdural strip/grid electrodes Needle electrodes Inserted through the skin for EMG (concentric or monopolar) and intraoperative monitoring Microelectrodes Tip diameter 0.5-5 μm, to record from inside a single cell; either metal (tungsten or platinum-iridium, etched and insulated except at the tip) or glass micropipettes filled with 3 M KCl.

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They have very high impedance (megohms to hundreds of megohms), so they demand an amplifier of extremely high input impedance, usually a unity-gain FET buffer close to the tip; they are also noisy and fragile Internal and catheter-tip electrodes Intracardiac, oesophageal, pacing and defibrillation electrodes Stimulating electrodes Deliver current rather than record — pacing, defibrillation, TENS, functional electrical stimulation Physiological Transducers • A transducer converts one form of energy into another — for measurement, usually a physical variable into an electrical signal.

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Active (self-generating) transducers produce their own output and need no external excitation — piezoelectric, thermocouple, photovoltaic and electromagnetic/induction types.

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Passive (modulating) transducers require an external excitation supply and vary a passive parameter — resistive (strain gauge, potentiometer, thermistor, RTD, photoresistor), capacitive and inductive (LVDT) types. • Displacement, position and motion: the resistive potentiometer (simple, robust, large output, but with friction and wear); the strain gauge, whose gauge factor = (ΔR/R)/(ΔL/L) is about 2 for metal foil and 50-200 for semiconductor types, used in a Wheatstone bridge with temperature compensation; the LVDT (linear variable differential transformer) — a primary with two opposed secondaries and a movable core, giving frictionless, high-resolution, essentially infinite-life displacement measurement with a phase-sensitive detector to indicate direction; capacitive transducers (C = εA/d); and for motion, accelerometers (piezoelectric or MEMS), tachometers and optical/magnetic encoders. • Pressure transducers: the sensing element is a diaphragm, bellows, Bourdon tube or capsule whose deflection is measured by a strain gauge, LVDT, capacitive or piezoresistive element.

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Direct (invasive) measurement uses a fluid-filled catheter coupled to an external transducer — cheap and re-zeroable but with damping, resonance and air-bubble problems, and the transducer must be levelled at the phlebostatic axis — or a catheter-tip (micromanometer) transducer, with a far better frequency response but at higher cost and fragility.

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Indirect measurement is by sphygmomanometry (5.2).

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Important adjuncts are zeroing to atmosphere, flushing systems and the square-wave damping test. • Photoelectric transducers: photovoltaic cells (generate a voltage — active), photoconductive cells/LDRs (resistance falls with light — passive), photodiodes (fast, linear, used in photovoltaic or reverse-biased photoconductive mode), phototransistors (more sensitive, slower) and photomultiplier tubes (extremely sensitive to very low light, used in gamma cameras, flow cytometers and luminometers).

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Their medical applications are the basis of pulse oximetry, photoplethysmography, colorimetry and spectrophotometry, blood cell counters, bilirubinometers and optical fibre sensors. • Other transducers: thermistor (a semiconductor with a large negative temperature coefficient — very sensitive but non-linear, the usual clinical temperature sensor), RTD/platinum resistance thermometer (linear and accurate, used as a standard), thermocouple (Seebeck effect, active, small and fast, needing a reference junction), piezoelectric (ultrasound transducers, phonocardiography, force), electromagnetic flow transducers and chemical/biosensors.

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Biomedical Recorders:

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ECG, EEG and EMG • Common front end: all three require an instrumentation amplifier built from three op-amps, with very high input impedance (> 10 MΩ, and far higher for microelectrodes), high differential gain, low noise and drift, and a very high common-mode rejection ratio (> 100 dB, i.e. > 10⁵:1) to reject the mains interference that is common to both inputs.

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This is followed by isolation (optical or transformer) for patient safety, a defibrillator protection circuit, filters, an ADC and the display or recorder.

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The driven right-leg circuit feeds the inverted common-mode signal back to the patient, further improving rejection. • Electrocardiograph (ECG): records the electrical activity of the heart (1.4).

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12 leads — three bipolar limb leads I, II and III (Einthoven's triangle, where lead II = lead I + lead III), three augmented unipolar limb leads aVR, aVL and aVF derived against Wilson's central terminal, and six unipolar chest leads V1-V6.

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Standard settings are a sensitivity of 10 mm/mV and a paper speed of 25 mm/s, so one small (1 mm) square is 0.04 s and one large (5 mm) square 0.2 s.

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Waves and normal intervals:

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P (atrial depolarisation), QRS (ventricular depolarisation, < 0.12 s), T (ventricular repolarisation), PR interval 0.12-0.20 s, QT interval.

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Uses: arrhythmia, ischaemia and infarction, chamber hypertrophy, electrolyte disturbance and drug effects.

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Artefacts: mains interference, baseline wander from respiration and motion, muscle tremor, loose electrodes and lead reversal. • Electroencephalograph (EEG): records cortical activity from scalp electrodes placed by the international 10-20 system, in which electrode spacing is 10 % or 20 % of the distance between the landmarks nasion, inion and the preauricular points, with odd numbers on the left, even on the right and z for the midline.

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Signals are only 10-100 μV, so amplification of about 10⁶ and meticulous technique are needed; recording may be bipolar or referential (monopolar), in montages.

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Rhythms: delta < 4 Hz (deep sleep, pathological when awake), theta 4-8 Hz (drowsiness, children), alpha 8-13 Hz (relaxed wakefulness with eyes closed, maximal occipitally and abolished by eye opening), beta 13-30 Hz (alert, mental activity, drug effect) and gamma > 30 Hz.

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Uses: epilepsy (the principal indication), encephalopathy, sleep studies, coma and brain-death assessment, depth of anaesthesia and evoked potentials.

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Artefacts: eye blink and movement, ECG, EMG from scalp muscles, sweat, electrode pop and mains. • Electromyograph (EMG): records the electrical activity of skeletal muscle, using surface electrodes (non-invasive, for kinesiology, biofeedback, prosthesis control and gait analysis, but with cross-talk and poor selectivity) or needle electrodes (concentric or monopolar, for clinical diagnosis, recording individual motor unit action potentials).

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Signals are 0.1-5 mV over 10-2,000 Hz, are inherently stochastic and biphasic, and are assessed at rest (where spontaneous fibrillation potentials indicate denervation), on minimal contraction (motor unit morphology — small short polyphasic units in myopathy, large long units in neuropathy) and on maximal effort (recruitment and interference pattern).

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EMG is normally accompanied by nerve conduction studies, which measure conduction velocity = distance ÷ (proximal latency − distal latency), normally about 50-60 m/s in large motor nerves, reduced in demyelination while amplitude falls in axonal loss.

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This section covers the concept of patient monitoring, bedside and central monitors, the measurement of heart rate, pulse rate, blood pressure, temperature and respiration, arrhythmia and ambulatory monitoring, fetal monitoring, pulse oximetry and blood flowmeters.

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System Concept • A patient monitoring system continuously measures one or more physiological variables, displays them, compares them with preset limits and raises an alarm when they are exceeded — its purpose being to detect deterioration early, to guide therapy and to record trends, not to treat.

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It is used in intensive and coronary care, operating theatres, recovery, emergency and high-dependency areas. • Architecture: sensors and electrodes → signal conditioning and isolation → processing → bedside monitor → network → central monitoring station, with printers, recorders and links to the electronic record. • The standard monitored parameters are ECG with heart rate and arrhythmia analysis, non-invasive blood pressure, SpO₂, respiration rate and temperature (together the 'vital signs'), extended in critical care by invasive arterial, central venous and pulmonary artery pressures, capnography (EtCO₂), cardiac output and intracranial pressure. • Alarms may be physiological (limit violations) or technical (lead off, sensor failure, low battery) and are graded by priority.

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The dominant practical problem is alarm fatigue from excessive false alarms, addressed by sensible limit setting, delays, and multi-parameter intelligent alarms. • Bedside monitors are modular, with parameter modules that can be transferred with the patient; central monitors display many beds at once with full disclosure recording, remote alarm and trending.

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Essential requirements are patient isolation and defibrillator protection, continuous operation, battery backup, infection control and ease of cleaning.

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Heart Rate and Pulse Rate • Heart rate is derived electrically from the ECG by detecting successive R waves and computing HR = 60 / RR interval (s), either beat to beat (instantaneous) or by averaging over several beats.

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A monitor must reject T waves, tall P waves, pacemaker spikes, muscle artefact and the mains, which is why QRS detectors use bandpass filtering around 10-25 Hz, differentiation, squaring and an adaptive threshold (the classic Pan-Tompkins algorithm), with a refractory blanking period of about 200 ms to prevent double counting. • Pulse rate is derived mechanically or optically from the peripheral pulse — by photoplethysmography (the pulse oximeter probe), by the arterial line waveform or by palpation.

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It equals the heart rate only when every electrical beat produces an effective mechanical contraction; a pulse deficit (pulse rate lower than heart rate) indicates ineffective beats, as in atrial fibrillation, frequent ectopics or pulseless electrical activity — a point examiners like.

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Blood Pressure Measurement • Non-invasive (indirect): the auscultatory method uses a sphygmomanometer cuff and stethoscope, systolic pressure being read at the appearance of Korotkoff phase I sounds and diastolic at their disappearance (phase V).

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Accuracy demands a cuff whose bladder width is about 40 % and length about 80 % of the arm circumference — a cuff that is too narrow reads falsely high and one too wide falsely low — with the arm at heart level and a deflation rate of 2-3 mmHg/s.

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The palpatory method gives systolic pressure only. • Automatic monitors use the oscillometric method: the cuff is inflated above systolic and deflated in steps while the amplitude of the pressure oscillations transmitted from the artery is measured.

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The point of maximum oscillation corresponds to the mean arterial pressure, which the device measures most reliably; systolic and diastolic are then derived by proprietary algorithms from fixed fractions of that maximum — which is why oscillometric systolic and diastolic values are less reliable than the mean, especially in arrhythmia, shock and severe hypertension.

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Other approaches are ultrasonic/Doppler detection of wall motion (useful in infants and in shock), finger volume-clamp (Peñaz) continuous monitors and tonometry. • Invasive (direct) arterial monitoring: a cannula in the radial (or femoral) artery is connected by short, stiff, fluid-filled tubing to an external strain-gauge transducer, with a continuous heparinised flush.

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It gives continuous beat-to-beat pressure, the waveform itself and arterial blood sampling.

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Its engineering problems are the dynamic response of the fluid-filled system: the system has a natural frequency (which should be well above the highest significant harmonic of the pressure wave, ideally > 20-25 Hz) and a damping ratio (optimally about 0.6-0.7).

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Under-damping — from long compliant tubing or air bubbles — causes resonance and an artificially high systolic reading; over-damping — from clot, kinks or a loose connection — blunts the waveform and underestimates systolic pressure.

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The system is checked by the fast-flush (square-wave) test and must be zeroed to atmosphere at the phlebostatic axis.

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Temperature and Respiration • Temperature sensors: the thermistor (large negative temperature coefficient, very sensitive, small, fast, but non-linear and requiring linearisation — the usual clinical probe), the RTD/platinum element (linear and stable, used as a reference), the thermocouple (small, fast, self-generating) and the infrared (tympanic and temporal) thermometer, which measures emitted radiation and gives a reading in a second or two.

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Sites: core temperature is best represented by pulmonary artery, oesophageal, nasopharyngeal, tympanic and rectal measurement, while oral, axillary and skin readings are progressively less reliable.

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Mercury glass thermometers have been withdrawn on environmental and safety grounds. • Respiration rate is most often obtained by transthoracic impedance pneumography, in which a small high-frequency current (typically 20-100 kHz, tens of microamperes) is passed between the ECG electrodes and the impedance variation with lung inflation is measured — convenient because it needs no extra sensor, but liable to motion and cardiogenic artefact and, critically, unable to detect obstructive apnoea, because chest wall movement continues without airflow.

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Alternatives are the thermistor or thermocouple at the nostril (sensing the temperature of expired air), capnography (which measures actual expired CO₂ and is the definitive monitor of ventilation, giving the end-tidal CO₂ value and waveform), strain-gauge or inductance belts, pneumotachography and acoustic methods.

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Arrhythmia and Ambulatory Monitoring • Cardiac arrhythmias are disturbances of rate, rhythm or conduction, classified by origin (sinus, atrial, junctional, ventricular) and by rate (brady- or tachyarrhythmia).

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Those that monitors must detect include ventricular fibrillation and ventricular tachycardia (the lethal ones), asystole, ventricular ectopics and their patterns (bigeminy, couplets, R-on-T), atrial fibrillation and flutter, supraventricular tachycardia, and the degrees of atrioventricular block. • The arrhythmia monitor works by QRS detection → feature extraction (RR interval, QRS width, amplitude, morphology and area) → template matching and classification into normal, ventricular ectopic, artefact or unclassified → rhythm analysis over successive beats → alarm.

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Its performance is judged by sensitivity and positive predictivity, and the design tension is between missing a true event and generating false alarms. • QRS detection techniques: threshold on amplitude (simplest, poor); first and second derivative methods exploiting the steep slope of the QRS; digital bandpass filtering (about 10-25 Hz) followed by differentiation, squaring and moving-window integration — the Pan-Tompkins algorithm, the standard; template matching/correlation; and wavelet, neural-network and deep-learning methods.

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All use a refractory period to avoid double detection and adaptive thresholds to follow changing amplitude. • Exercise stress testing records the ECG, blood pressure and symptoms during graded exercise on a treadmill (Bruce protocol) or bicycle ergometer, to unmask ischaemia that is absent at rest.

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The diagnostic criterion is horizontal or downsloping ST-segment depression of ≥ 1 mm (0.1 mV) 80 ms after the J point; the test also assesses exercise capacity, chronotropic response and exercise-induced arrhythmia.

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It demands resuscitation facilities and has clear termination criteria.

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Technically it is the most artefact-prone ECG recording, requiring careful skin preparation, torso electrode placement and signal averaging. • Ambulatory monitoring: the Holter monitor records 24-48 hours of continuous ECG on a body-worn recorder for later analysis — used for intermittent palpitations, syncope, silent ischaemia and pacemaker assessment.

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For less frequent symptoms, patient-activated event recorders, external loop recorders, patch monitors, mobile cardiac telemetry and implantable loop recorders (up to about three years) are used.

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Ambulatory blood pressure monitoring over 24 hours identifies white-coat and masked hypertension and the nocturnal dipping pattern.

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Fetal Monitoring • The cardiotocograph (CTG) records the fetal heart rate and uterine contractions simultaneously on a common time axis, which is what makes it interpretable — the relation of the heart rate to the contraction is the whole point. • Methods of monitoring fetal heart rate: external (indirect) — Doppler ultrasound (1-2 MHz) transducer on the maternal abdomen, detecting the motion of the fetal heart valves and walls, with autocorrelation to derive beat-to-beat rate; also abdominal fetal ECG and phonocardiography.

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Internal (direct) — a fetal scalp electrode (spiral electrode) attached to the presenting part once the membranes have ruptured and the cervix is dilated, giving a true fetal ECG and therefore accurate beat-to-beat variability, at the cost of being invasive with a small risk of trauma and infection. • Monitoring labour activity: external tocodynamometry — a strain-gauge pressure transducer strapped to the abdominal wall, which reliably shows the frequency and duration of contractions but not their true intensity or the resting tone; and the intrauterine pressure catheter, which measures actual intra-amniotic pressure in mmHg and so gives intensity and baseline tone. • Interpretation rests on baseline rate (normal 110-160 beats/min), baseline variability (normally 5-25 beats/min — reduced variability being an important warning sign), accelerations (reassuring) and decelerations, which are classified as early (head compression, benign, mirroring the contraction), variable (cord compression) and late (uteroplacental insufficiency — the ominous pattern, beginning after the peak of the contraction).

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The recording system runs at a paper speed of 1, 2 or 3 cm/min, and modern systems add central display, archiving and computerised analysis.

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Supplementary tests are fetal scalp blood sampling and fetal pulse oximetry.

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Pulse Oximetry and Blood Flowmeters • Pulse oximetry measures arterial oxygen saturation (SpO₂) non-invasively and continuously, and rests on two principles.

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First, spectrophotometry (the Beer-Lambert law): oxyhaemoglobin and deoxyhaemoglobin have different absorption spectra, so measurement at two wavelengths — 660 nm (red), where deoxyhaemoglobin absorbs more, and 940 nm (infrared), where oxyhaemoglobin absorbs more — allows their ratio to be computed.

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Second, plethysmography: only arterial blood pulsates, so the device separates the pulsatile AC component from the constant DC component (tissue, venous and capillary blood) and computes the ratio of ratios R = (AC/DC)₆₆₀ ÷ (AC/DC)₉₄₀, which is converted to saturation by an empirical calibration curve derived from healthy volunteers.

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Construction: two LEDs and a photodiode, alternately pulsed at a few hundred hertz with a dark interval to subtract ambient light. • Limitations, which are heavily examined: carboxyhaemoglobin absorbs like oxyhaemoglobin at 660 nm, so the reading is falsely high in carbon monoxide poisoning; methaemoglobin drives the reading towards 85 %; poor perfusion, hypothermia, vasoconstriction and hypotension give an inadequate pulsatile signal; motion artefact; nail varnish, dyes (methylene blue) and strong ambient light; venous pulsation in tricuspid regurgitation; and the flatness of the upper part of the oxyhaemoglobin dissociation curve, which means SpO₂ falls only late as PaO₂ drops.

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Anaemia does not affect saturation, only content; and pulse oximetry says nothing about ventilation or PaCO₂, for which capnography is required. • Electromagnetic blood flowmeter: based on Faraday's law of induction.

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Blood, being an electrolytic conductor, moving with velocity v through a magnetic field B in a vessel of diameter d, generates a voltage E = B·L·v across electrodes at right angles to both, so the induced voltage is directly proportional to the mean flow velocity and hence to volume flow.

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A perivascular cuff probe is placed around an exposed vessel, so the method is invasive and used surgically or experimentally; alternating (sine or square wave) excitation is used rather than DC to avoid polarisation and baseline drift.

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It measures flow in absolute units without entering the vessel and is insensitive to the velocity profile, but requires surgical exposure and careful zero setting. • Ultrasonic blood flowmeter: either transit-time, in which the difference in propagation time upstream and downstream is proportional to velocity, or — far more commonly in clinical use — Doppler, in which the frequency shift of ultrasound reflected from moving red cells gives velocity by Δf = 2f₀v cos θ / c (4.4).

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Doppler flowmeters may be continuous wave or pulsed, and combined with a measured vessel cross-section give volume flow.

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They are non-invasive and widely available, but are angle-dependent and their accuracy suffers from uncertainty in the vessel diameter and velocity profile.

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This section covers wireless biomedical telemetry, single- and multi-channel systems, multi-patient and implantable telemetry, the transmission of analogue physiological signals, and telemedicine.

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Principles of Biomedical Telemetry • Biotelemetry is the measurement of a physiological variable at a distance from the subject, the signal being transmitted without a direct physical (wired) connection. • Why it is used: to allow the patient to move freely (ambulatory and exercise monitoring, rehabilitation); to eliminate the electrical connection between patient and mains-powered equipment, which is a major safety advantage (no leakage-current path); to monitor in situations where cables are impossible (athletes, animals, space flight, battlefield and disaster); to allow monitoring during transport; and to reduce cable clutter and cross-infection. • The generic system: transducer/electrode → signal conditioning → modulator → transmitter and antenna → radio link → receiving antenna → receiver → demodulator → signal processing → display/recording, with a battery power supply at the patient end. • Design requirements at the patient end: small, light and comfortable; very low power consumption for long battery life; patient safety and isolation; adequate range; immunity to interference and to fading; and regulatory compliance with allocated frequency bands — medical telemetry uses designated bands such as the WMTS, ISM bands and, at short range, Bluetooth Low Energy, Wi-Fi, Zigbee and MICS (402-405 MHz for implants).

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Modulation and Transmission of Analogue Physiological Signals • Physiological signals are of very low frequency (DC to a few hundred hertz) and cannot be radiated directly, since the antenna would have to be impossibly large; they must therefore modulate a radio-frequency carrier. • Modulation schemes:

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AM (simple but very susceptible to noise and amplitude fading — little used);

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FM (signal amplitude carried as frequency deviation; far better noise immunity, and the standard analogue method, usually as FM/FM — the signal first frequency-modulates a subcarrier, which then frequency-modulates the main carrier);

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PM; the pulse modulations — PAM, PDM/PWM, PPM and PCM; and digital schemes (FSK, PSK, QPSK) with error detection and correction, which now dominate. • Why digital telemetry has displaced analogue: noise immunity and regeneration, error detection and correction, encryption and privacy, easy multiplexing of many channels and many patients, compression, and direct integration with computers and networks. • Multiplexing allows several signals to share one carrier: frequency division multiplexing (FDM), in which each signal modulates its own subcarrier and the subcarriers are summed — the classic analogue approach; and time division multiplexing (TDM), in which the channels are sampled in turn and interleaved, with a synchronising/frame word so that the receiver can sort them out — the natural digital approach.

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Code division multiple access is also used. • Sampling must obey the Nyquist criterion — the sampling rate must be at least twice the highest frequency present — with an anti-aliasing low-pass filter before the sampler; in practice ECG is sampled at 250-500 Hz (up to 1 kHz for diagnostic work) and EEG at 250-500 Hz.

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Single-Channel, Multi-Channel and Multi-Patient Systems System Description Single-channel telemetry One physiological variable, usually ECG, from one patient.

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The patient wears a small battery transmitter with electrodes; the signal frequency-modulates a subcarrier and then the carrier.

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Simple, cheap, long battery life — the classic coronary care ambulatory ECG transmitter.

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A single-channel link can also carry temperature or respiration Multi-channel telemetry Several variables from one patient (for example ECG, respiration and temperature) sent over one radio link by FDM with separate subcarriers, or by TDM/PCM after multiplexing and digitising.

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Needs more bandwidth and power; channel crosstalk and synchronisation must be managed Multi-patient telemetry Many patients monitored simultaneously at a central station.

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Each patient's transmitter is assigned a separate carrier frequency (or a separate time slot or code), and the receiving system has matching receivers or a scanning receiver feeding the central monitor.

14

Requires careful frequency planning, adequate channel separation, antenna distribution around the ward, and management of interference and dropout — typically used in a step-down or telemetry ward where patients are ambulant Implantable telemetry Transmitter implanted within the body — see below Implantable Telemetry • Implantable telemetry systems transmit data from a device or sensor inside the body: pacemakers and ICDs (interrogation, programming and remote follow-up), implantable loop recorders, intracranial and intraocular pressure sensors, continuous glucose monitors, neurostimulators, orthopaedic implants with instrumented sensors, and ingestible capsule endoscopes and pH capsules. • Special constraints: hermetic biocompatible encapsulation (titanium, ceramic or glass); extremely low power consumption, since the battery cannot easily be replaced; size and shape; attenuation of radio waves by tissue, which increases sharply with frequency and so restricts implants to low frequencies and short range; heating limits; and security, since a device that can be programmed wirelessly can in principle be attacked. • Powering: a primary battery (lithium-iodine in pacemakers, 7-12 years), inductive coupling through a pair of coils (which also carries data, the standard method for cochlear implants and for recharging), radiofrequency energy harvesting, and energy scavenging from motion, heat or biochemical sources. • Communication: historically inductive/near-field at low frequency with a wand held over the device; now increasingly the MICS band (402-405 MHz) or Bluetooth Low Energy, giving a few metres of range, which enables remote home monitoring of pacemakers and ICDs with automatic daily transmission to the clinic.

15

Telemedicine • Telemedicine is the delivery of health care at a distance using information and communication technology, when distance is a critical factor; telehealth is the broader term including education, administration and public health. • Modes: store-and-forward (asynchronous) — images, ECGs or records are captured and sent for later reporting, which suits teleradiology, teledermatology, telepathology and teleophthalmology and needs no simultaneous presence; real-time (synchronous) — live video consultation, teleconsultation and telesurgery, requiring adequate bandwidth at both ends; and remote patient monitoring — home transmission of vital signs, glucose, weight and device data for chronic disease and post-discharge care.

16

Mobile health (mHealth) uses phones and wearables. • Components: acquisition devices (cameras, digital stethoscopes, ECG, ultrasound probes, otoscopes), a communication link (broadband, mobile data, VSAT/satellite for remote areas), standards (DICOM, HL7), secure servers and storage, and the clinical software and workflow. • Benefits: access to specialist care in remote and mountainous areas — the central argument in Nepal, where geography, limited road access and the concentration of specialists in Kathmandu make physical referral slow and expensive; earlier diagnosis; reduced travel cost and time; support and continuing education for rural health workers; better use of scarce specialists; and continuity of care.

17

Nepal's national telemedicine programme linking district hospitals to central referral centres is the standing example. • Challenges: connectivity, bandwidth and reliable electricity; equipment cost and maintenance; training and acceptance by staff and patients; data security, privacy and confidentiality; medico-legal questions of licensing, liability, consent and record-keeping; reimbursement; the impossibility of physical examination; and the need for standards and interoperability.

1

This section covers the measurement of cardiac output by indicator dilution, dye dilution, thermodilution, arterial pressure waveform analysis, impedance and ultrasound, together with pulmonary function measurement by spirometry and the measurement of lung volumes.

2

Definitions and the Fick Principle • Cardiac output CO = heart rate × stroke volume ≈ 5 L/min at rest (1.4); the cardiac index is CO divided by body surface area, about 2.5-4.0 L/min/m².

3

Its measurement matters in shock, heart failure, sepsis, major surgery and in the assessment of valve and congenital disease. • The Fick principle underlies all of these methods: the total uptake or release of a substance by an organ is the product of the blood flow and the arteriovenous concentration difference.

4

Applied to oxygen in the lungs, CO = VO₂ / (CaO₂ − CvO₂) — the oxygen consumption divided by the arteriovenous oxygen content difference.

5

It is the reference standard, but requires a mixed venous sample from the pulmonary artery and a measurement of oxygen consumption, which makes it cumbersome.

6

Indicator Dilution Methods • Principle: a known quantity of an indicator is injected upstream and its concentration measured downstream as a function of time; the more rapidly it is diluted and washed through, the greater the flow.

7

This is expressed by the Stewart-Hamilton equation:

8

CO = m / ∫C(t) dt — the mass of indicator injected divided by the area under the concentration-time curve. • Requirements of an ideal indicator: non-toxic, well mixed with the blood, not lost from the circulation or metabolised during the first pass, easily and accurately measured, and eliminated reasonably quickly so that the measurement can be repeated. • Dye dilution: the classic method, using indocyanine green (absorption peak about 805 nm, the isosbestic point of haemoglobin, so the reading is independent of oxygen saturation) injected into a central vein and sampled from a peripheral artery through a cuvette densitometer.

9

Its characteristic problem is recirculation: the descending limb of the curve is interrupted by indicator returning for a second pass, so the true curve must be recovered by semilogarithmic extrapolation of the exponential downslope before the area is computed.

10

Dye also accumulates with repeated measurements. • Thermodilution is the standard clinical method and uses cold (or room-temperature) saline as the indicator and temperature as the measured variable.

11

A Swan-Ganz (pulmonary artery) catheter carries a proximal port in the right atrium and a thermistor at the tip in the pulmonary artery;

12

10 mL of cold saline is injected and the resulting transient fall in pulmonary artery blood temperature is recorded, the computer integrating the temperature-time curve.

13

Advantages: no arterial sampling, no dye accumulation, and negligible recirculation because the cold is dissipated, so measurements can be repeated every few minutes; the same catheter also gives pulmonary artery and wedge pressures and mixed venous saturation.

14

Sources of error: tricuspid regurgitation, intracardiac shunts, arrhythmia, rapid intravenous infusion, incorrect injectate volume or temperature, slow or uneven injection, and catheter malposition; the accepted practice is to average three injections within 10 %.

15

Invasiveness and complications (arrhythmia, infection, pulmonary artery rupture) have reduced its use in favour of less invasive methods. • Lithium dilution and transpulmonary thermodilution (PiCCO) use a peripheral vein and an arterial catheter instead of a pulmonary artery catheter, and also provide calibration for pulse contour analysis.

16

Continuous Cardiac Output from the Arterial Pressure Waveform • Pulse contour (pulse power) analysis derives stroke volume beat by beat from the shape of the arterial pressure waveform.

17

The physiological basis is the Windkessel model: the area under the systolic portion of the pressure curve is proportional to stroke volume, the constant of proportionality depending on aortic compliance and systemic vascular resistance. • Calibrated systems (PiCCO with transpulmonary thermodilution, LiDCO with lithium dilution) determine that constant by an independent dilution measurement and then track changes continuously.

18

Uncalibrated systems (FloTrac and similar) estimate compliance from the patient's age, sex and body size and from waveform characteristics, and need only an existing arterial line. • Advantages: continuous, beat-to-beat output with no pulmonary artery catheter, and derived indices of fluid responsiveness — stroke volume variation and pulse pressure variation — which predict whether a patient will respond to a fluid challenge.

19

Limitations: dependence on a high-quality, correctly damped arterial trace (an over- or under-damped line invalidates it), inaccuracy in arrhythmia, aortic regurgitation, intra-aortic balloon counterpulsation and rapidly changing vascular tone, and drift requiring recalibration.

20

Impedance and Ultrasound Methods • Impedance cardiography (thoracic electrical bioimpedance) is entirely non-invasive.

21

A small high-frequency alternating current (about 20-100 kHz, a few milliamperes) is passed through the thorax between outer electrodes, and the resulting voltage is sensed by inner electrodes.

22

Because blood is the best conductor in the thorax, thoracic impedance falls as the aorta fills with each systole.

23

The maximum rate of change of impedance, dZ/dtmax, together with the ventricular ejection time and the baseline impedance, is used in equations of the Kubicek or Sramek-Bernstein type to compute stroke volume.

24

Its advantages are that it is non-invasive, continuous, cheap and repeatable; its limitations are sensitivity to electrode placement, motion, arrhythmia, and above all to pulmonary oedema, pleural fluid and obesity, which change thoracic impedance for non-cardiac reasons, so its absolute accuracy is limited even though trends are useful.

25

Bioreactance (measuring phase shift rather than amplitude) is a more robust development. • Ultrasound methods:

26

Doppler echocardiography computes stroke volume = velocity-time integral (VTI) × cross-sectional area of the left ventricular outflow tract, the VTI being obtained from the pulsed Doppler trace and the area from the measured LVOT diameter (area = πd²/4);

27

It is non-invasive, gives simultaneous structural and valvular information, and is now the commonest clinical approach — but it is operator-dependent, intermittent, and very sensitive to the LVOT diameter measurement, since that is squared.

28

Oesophageal Doppler uses a probe in the oesophagus aimed at the descending aorta, giving a continuous signal well suited to intraoperative optimisation, with the aortic area taken from a nomogram or measured.

29

Transit-time flow probes are used on vessels during surgery. • Other methods: the partial CO₂ rebreathing (NICO) technique, which applies the Fick principle to carbon dioxide, and cardiac MRI phase-contrast flow mapping, which is the current non-invasive reference standard.

30

Pulmonary Function Measurement:

31

Spirometry • Spirometry measures the volume and flow of air moved by the lungs.

32

Instruments are either volume-displacement spirometers — the classic water-sealed bell, the dry rolling-seal and the wedge/bellows types — or, now almost universally, flow-sensing (pneumotachograph) spirometers, which measure flow and integrate it to obtain volume.

33

Flow sensors work by the pressure drop across a fixed resistance (Fleisch capillary or Lilly screen type, obeying Poiseuille's law), by a heated-wire (thermal) anemometer, by a turbine/rotating vane, or by an ultrasonic transit-time sensor. • Procedure and quality: the patient performs a maximal inspiration followed by a maximal forced expiration through a mouthpiece with a nose clip, seated upright; at least three acceptable and two reproducible manoeuvres are required, with acceptability judged by a sharp start (back-extrapolated volume), no cough or leak, and a plateau at the end.

34

Results are corrected to BTPS and interpreted against predicted values for age, height, sex and ethnicity. • The measurements:

35

FVC (forced vital capacity), FEV₁ (forced expiratory volume in the first second), the FEV₁/FVC ratio, PEFR (peak expiratory flow rate), FEF₂₅₋₇₅ (mid-expiratory flow, sensitive to small airway disease), MVV and the flow-volume loop, whose shape is diagnostic — a concave (scooped) expiratory limb in obstruction, a small narrow loop in restriction, and flattening of the inspiratory or expiratory limb in extrathoracic or intrathoracic upper airway obstruction.

36

Pattern FEV₁ FVC FEV₁/FVC Examples Obstructive ↓↓ Normal or ↓ Reduced (< 0.7) Asthma (reversible with bronchodilator), COPD, bronchiectasis Restrictive ↓ ↓↓ Normal or increased Pulmonary fibrosis, kyphoscoliosis, obesity, neuromuscular disease, pleural disease Mixed ↓↓ ↓ Reduced Combined disease Measurement of Lung Volumes • As set out in 1.4, the volumes are TV 500 mL, IRV about 3,000 mL, ERV about 1,100 mL and RV about 1,200 mL, and the capacities IC = TV + IRV, FRC = ERV + RV, VC = TV + IRV + ERV ≈ 4,600 mL and TLC = VC + RV ≈ 5,800 mL. • The essential point: spirometry can measure only the air that moves through the mouth, so residual volume — and therefore FRC and TLC — cannot be measured by spirometry and requires one of three techniques. • Helium dilution (closed circuit): the patient rebreathes from a spirometer containing a known volume and concentration of helium, an insoluble inert gas; helium is diluted into the lung gas until equilibrium, and from C₁V₁ = C₂(V₁ + V₂) the unknown lung volume is calculated.

37

It is simple, but underestimates volume in severe obstruction because poorly ventilated and trapped gas does not communicate. • Nitrogen washout (open circuit): the patient breathes 100 % oxygen and all the exhaled nitrogen is collected; knowing that alveolar gas was initially about 79-80 % nitrogen, the original lung volume follows.

38

It shares the same limitation with trapped gas. • Body plethysmography is the reference method: the patient sits in a sealed box and pants against a closed shutter, and Boyle's law (P₁V₁ = P₂V₂) applied to the compression and decompression of thoracic gas gives the thoracic gas volume.

39

Because it measures all compressible gas in the chest, including trapped gas, it is the most accurate — and comparison of plethysmographic with dilutional volume quantifies air trapping. • Other pulmonary function tests: the diffusing capacity for carbon monoxide (DLCO), single-breath, which assesses the alveolar-capillary membrane and is reduced in emphysema and fibrosis and raised in pulmonary haemorrhage; peak flow meters for asthma self-monitoring; bronchial provocation and reversibility testing; arterial blood gases (5.5); and pulse oximetry (5.2).

1

This section covers biochemistry analysers and colorimeters, electrolyte analysers, the microscope, the centrifuge, ELISA readers and washers, biosafety cabinets and autoclaves, blood gas analysers and blood cell counters including the Coulter principle.

2

Colorimeters, Spectrophotometers and Biochemistry Analysers • Principle: almost all clinical biochemistry rests on photometry, governed by the Beer-Lambert law — A = ε·c·l, where absorbance A = log₁₀(I₀/I), ε is the molar absorptivity, c the concentration and l the path length (usually 1 cm).

3

Absorbance is directly proportional to concentration, while transmittance is logarithmically related — which is why absorbance, not transmittance, is used for quantification.

4

The law fails at high concentrations and with stray light or non-monochromatic light. • Components of a photometer, in order: light source (tungsten filament for the visible range, deuterium for ultraviolet, LED in modern compact instruments), monochromator — a filter in a colorimeter (cheap, visible range only, broad bandpass) or a prism or diffraction grating in a spectrophotometer (continuously selectable wavelength, narrow bandpass, ultraviolet capability), cuvette/sample holder (glass or plastic for visible, quartz for ultraviolet), detector (photodiode, phototube or photomultiplier) and readout.

5

The filter chosen is complementary in colour to the solution — a blue solution is read with a red filter, because the solution absorbs most strongly the colour it does not transmit. • Biochemistry (clinical chemistry) analysers automate this: sample and reagent pipetting, mixing, incubation at 37 °C, photometric reading and calculation.

6

They are classified as semi-automatic (manual pipetting, automatic reading) or fully automatic, and as batch, random-access (any test on any sample in any order — the modern standard) or continuous-flow analysers.

7

Measurement modes are end-point (read after the reaction is complete — glucose, urea, protein), kinetic/rate (the rate of absorbance change — enzymes such as ALT, AST, ALP), fixed-time and turbidimetric or nephelometric (light scattering by immune complexes — CRP, immunoglobulins).

8

Common analytes are glucose, urea, creatinine, bilirubin, total protein and albumin, cholesterol and triglycerides, uric acid, calcium, and the enzymes. • Quality control is inseparable from the instrument: calibration with standards, daily internal QC at two or three levels plotted on Levey-Jennings charts with Westgard rules, external quality assessment, reagent and cuvette maintenance, and the control of interference from haemolysis, lipaemia and icterus.

9

Electrolyte Analysers and Blood Gas Analysers • Electrolyte analysers measure Na⁺, K⁺, Cl⁻, ionised Ca²⁺ and sometimes Li⁺ by ion-selective electrodes (ISE).

10

An ISE is a potentiometric sensor: a membrane selective for one ion develops a potential relative to a reference electrode that follows the Nernst equation, E = E₀ + (RT/nF)·ln(activity), so the potential is proportional to the logarithm of ion activity — about 59 mV per decade for a monovalent ion at 25 °C.

11

Membranes: glass for Na⁺ and H⁺, valinomycin in PVC for K⁺, and ion-exchanger membranes for Cl⁻ and Ca²⁺.

12

Measurement may be direct (undiluted sample — measures true activity and is unaffected by protein or lipid) or indirect (diluted — subject to pseudohyponatraemia in hyperlipidaemia and hyperproteinaemia).

13

The older method was flame photometry, in which sodium, potassium and lithium emit characteristic wavelengths (Na 589 nm yellow, K 766 nm violet) when aspirated into a flame. • Blood gas analysers measure pH, PCO₂ and PO₂ directly on a heparinised anaerobic arterial sample, and calculate bicarbonate, base excess and saturation.

14

Three classic electrodes: the pH electrode, a potentiometric glass membrane electrode against a calomel or Ag/AgCl reference; the Severinghaus PCO₂ electrode, a pH electrode behind a CO₂-permeable silicone/Teflon membrane and a bicarbonate solution — CO₂ diffuses in, forms carbonic acid and changes the pH, which is measured, so it is potentiometric and indirect; and the Clark PO₂ electrode, an amperometric sensor with a platinum cathode and Ag/AgCl anode behind an oxygen-permeable membrane, polarised at about −0.6 V, in which oxygen is reduced at the cathode and the resulting current is proportional to PO₂.

15

Modern analysers add ISEs for electrolytes, glucose and lactate and a co-oximeter for haemoglobin fractions.

16

Pre-analytical errors dominate: air bubbles (falsely raising PO₂ and lowering PCO₂), excess heparin dilution, delay without cooling (cells continue to metabolise), and venous rather than arterial sampling.

17

The instrument requires regular calibration with tonometered gases and buffers, and control of protein deposition on the membranes.

18

Microscope and Centrifuge • The compound light microscope: total magnification = objective × eyepiece (typically 10× eyepiece with 10×, 40× and 100× oil-immersion objectives, giving 100× to 1,000×).

19

Resolution, the ability to distinguish two close points, is given by d = 0.61λ / NA (or the Abbe limit λ/2NA), where NA = n·sin θ is the numerical aperture — hence the use of immersion oil (n ≈ 1.5) to raise NA above 1 and improve resolution, and the limit of about 0.2 μm for visible light.

20

Köhler illumination with correct condenser and iris adjustment is essential; closing the iris increases contrast but degrades resolution. • Types: bright field (routine, stained specimens), dark field (unstained motile organisms such as spirochaetes), phase contrast (unstained living cells, converting phase differences into amplitude differences), fluorescence (immunofluorescence, auramine staining for tuberculosis), polarising (crystals — urate and pyrophosphate in joint fluid), inverted (cell culture) and electron (TEM and SEM, with resolution in nanometres). • The centrifuge separates particles of different density by sedimentation in a centrifugal field.

21

The important quantity is not speed but the relative centrifugal force, RCF (g) = 1.118 × 10⁻⁵ × r × N², with r the radius in centimetres and N the speed in rpm — so the radius of the rotor matters as much as the rpm, and protocols must specify g, not rpm.

22

Types: fixed-angle (faster, pellet on the side wall), swinging-bucket (horizontal during spin, flat pellet, better for gradients and blood bags), microcentrifuge, refrigerated (for heat-labile samples), haematocrit (high speed, capillary tubes), cytocentrifuge (cells onto a slide) and ultracentrifuge (up to 100,000 g, for subcellular fractionation).

23

Safety and practice: always balance opposing tubes by mass, never open the lid while the rotor is spinning, use sealed buckets or aerosol-tight rotors for infectious material, inspect rotors for corrosion and observe their service life, and never exceed the rated speed.

24

ELISA Reader and Washer, Biosafety Cabinet and Autoclave • ELISA (enzyme-linked immunosorbent assay) detects an antigen or antibody by an enzyme-labelled immunological reaction producing a colour whose intensity is proportional to the amount present.

25

Formats are direct, indirect (the commonest for antibody detection — HIV, hepatitis), sandwich (for antigen) and competitive (for small molecules).

26

The steps are coating → blocking → sample incubation → washing → conjugate (enzyme-labelled antibody, usually horseradish peroxidase or alkaline phosphatase) → washing → substrate (TMB or pNPP) → stop → read. • The ELISA washer dispenses and aspirates wash buffer from the 96-well plate several times between steps.

27

Its function is decisive: washing removes unbound reagent and is the single greatest determinant of background and of assay reliability, so blocked or misaligned needles, incomplete aspiration, cross-contamination between wells and drying of the plate are the classic causes of failure. • The ELISA reader (microplate photometer) is a filter photometer that reads the absorbance of each well of a 96-well plate, usually at 450 nm for TMB with a reference wavelength (620-650 nm) to correct for plate imperfections.

28

Software computes a standard curve for quantitative assays or compares with a cut-off value for qualitative ones. • Biosafety cabinet (BSC) protects against aerosols generated in microbiological work, by means of HEPA filters (99.97 % of particles of 0.3 μm) and controlled airflow.

29

Class I — inward airflow only: protects the operator and environment but not the sample.

30

Class II — inward airflow plus HEPA-filtered downflow over the work surface: protects operator, sample and environment, and is the standard cabinet in clinical and research laboratories (subtypes A1, A2, B1, B2).

31

Class III — a totally enclosed gas-tight glove box at negative pressure, for the most dangerous agents.

32

A biosafety cabinet is not a fume hood and a laminar flow (clean) bench is not a biosafety cabinet — the horizontal laminar flow bench blows filtered air towards the operator and protects only the product, so it must never be used with infectious material.

33

Cabinets must be certified on installation and annually, and the sash height and airflow alarms respected. • Autoclave: sterilisation by saturated steam under pressure, which kills by moist heat coagulating and denaturing proteins — far more effective than dry heat because water conducts heat and hydrated proteins denature at lower temperatures.

34

The standard cycles are 121 °C at 15 psi (103 kPa) for 15-20 minutes or 134 °C at 30 psi for 3-4 minutes.

35

Air must be completely displaced, since trapped air lowers the temperature at a given pressure and is the commonest cause of failure; hence downward-displacement and, better, pre-vacuum autoclaves.

36

Monitoring is by physical (temperature, pressure, time charts), chemical (autoclave tape — which shows only that the pack was processed;

37

Bowie-Dick test for air removal) and, definitively, biological indicators — spores of Geobacillus stearothermophilus.

38

Items must be correctly packed and not overloaded, and liquids cooled before opening.

39

Blood Cell Counters • Methods of cell counting: manual counting in a Neubauer haemocytometer with a diluting fluid (Turk's for WBC, Hayem's or ammonium oxalate for RBC and platelets) — cheap, needing no power, but slow, imprecise (10-20 % coefficient of variation) and dependent on the operator; and automated counting by the electrical impedance (Coulter) principle, by optical light scatter and flow cytometry, or by a combination with fluorescence. • The Coulter principle is the classic examinable topic.

40

Cells suspended in an electrolyte are drawn through a small aperture across which a constant current is maintained between two electrodes.

41

Each cell, being a poor conductor, displaces its own volume of electrolyte and momentarily increases the resistance across the aperture, producing a voltage pulse.

42

The number of pulses gives the cell count and the amplitude of each pulse is proportional to the cell volume, so a size distribution histogram is obtained — from which the MCV and the discrimination of platelets from red cells follow.

43

Sources of error: coincidence (two cells passing together, corrected electronically), aperture blockage, air bubbles and debris, and interference from nucleated red cells, cold agglutinins, lipaemia and giant platelets. • Modern analysers combine impedance with hydrodynamic focusing (which confines cells to the centre of the aperture and greatly improves precision), radiofrequency conductivity (giving information about internal structure and nuclear density), laser light scatter (forward scatter for size, side scatter for granularity), cytochemical staining and fluorescence, to produce the five-part differential white cell count and reticulocyte counts.

44

Haemoglobin is measured photometrically in a separate channel, classically by the cyanmethaemoglobin method read at 540 nm (now often cyanide-free).

45

The haematocrit is calculated as RBC × MCV, and the indices MCH = Hb/RBC and MCHC = Hb/HCT follow.

46

Flagged or abnormal samples must still be confirmed by microscopic examination of a stained blood film.

1

This section covers the basic audiometer, pure-tone audiometry by air and bone conduction, speech audiometry, Bekesy audiometry, calibration and the interpretation of the audiogram.

2

Basis in Hearing • As set out in 1.3, sound is collected by the external ear, amplified by the ossicular impedance-matching mechanism of the middle ear, and transduced by the hair cells of the organ of Corti on the tonotopically organised basilar membrane.

3

The audible range is about 20 Hz to 20 kHz, with greatest sensitivity at 1-4 kHz, and the reference threshold 0 dB SPL corresponds to a pressure of 20 μPa. • Decibels: dB SPL = 20 log₁₀(P/P₀).

4

Audiometry, however, uses dB HL (hearing level), in which 0 dB HL at every frequency is the average threshold of normal young adults — so the audiogram is a straight line at 0 dB for normal hearing, whereas the equivalent SPL differs greatly from one frequency to another. dB SL (sensation level) is measured above the individual's own threshold. • Types of hearing loss: conductive (external or middle ear — wax, perforation, effusion, otosclerosis: air conduction impaired, bone conduction normal, giving an air-bone gap); sensorineural (cochlea or auditory nerve — presbycusis, noise, ototoxicity, Ménière's: air and bone conduction equally reduced, no air-bone gap); and mixed.

5

Degrees are graded roughly as normal ≤ 25 dB, mild 26-40, moderate 41-55, moderately severe 56-70, severe 71-90 and profound > 90 dB HL.

6

The Basic Audiometer • An audiometer is an instrument that generates acoustic signals of accurately known frequency and intensity for the measurement of hearing thresholds. • Components: a signal generator (oscillator) producing pure tones at the standard audiometric frequencies 125, 250, 500, 1,000, 2,000, 4,000 and 8,000 Hz (with the inter-octaves 750, 1,500, 3,000 and 6,000 Hz); a frequency selector; an attenuator calibrated in 5 dB steps, typically from −10 to 110 dB HL; an interrupter/tone presenter switch with a rise-fall time that avoids audible clicks; an output selector routing the signal to the air-conduction earphones (supra-aural TDH or insert phones), the bone vibrator, or a loudspeaker for free-field testing; a masking noise generator (narrow-band noise for tones, speech noise for speech); a channel for speech input with a microphone or recorded material and a VU meter; and a patient response button. • Classification (IEC/ANSI types): type 4/screening audiometers (air conduction only, limited frequencies — for school and industrial screening), type 3 diagnostic, type 2 clinical and type 1 research audiometers with speech, masking and extended features. • Environment and calibration: testing requires a sound-treated room or booth meeting permissible ambient noise limits, since background noise raises apparent thresholds.

7

The audiometer must be listened to daily (biological check), and calibrated electroacoustically at least annually with a sound level meter and the appropriate coupler — a 6 cc coupler for supra-aural earphones, a 2 cc coupler for insert phones and an artificial mastoid for the bone vibrator.

8

An uncalibrated audiometer produces confidently wrong results, which is why calibration is an examinable topic in its own right.

9

Pure-Tone Audiometry • Air conduction testing presents the tone through earphones, so the signal traverses the whole auditory pathway — external, middle and inner ear and the nerve.

10

Bone conduction testing places a vibrator on the mastoid or forehead, transmitting vibration directly to the cochlea through the skull and bypassing the external and middle ear.

11

Comparing the two is what distinguishes conductive from sensorineural loss: an air-bone gap of more than 10-15 dB indicates a conductive component. • Procedure: begin with the better ear at 1,000 Hz, then test 1, 2, 4, 8 kHz followed by 500 and 250 Hz, and retest 1,000 Hz to check reliability.

12

Threshold is sought by the modified Hughson-Westlake 'down 10, up 5' method, and the threshold is defined as the lowest level at which the patient responds to at least 50 % (usually 2 out of 3) of presentations. • Masking is essential and frequently examined.

13

Because sound presented to one ear can cross the skull and be heard by the other (cross-hearing), a narrow-band masking noise is presented to the non-test ear so that only the test ear can respond.

14

Interaural attenuation — the loss in crossing — is about 40 dB for supra-aural earphones (up to 70 dB for insert phones) but essentially 0 dB for bone conduction, which is why bone conduction testing almost always requires masking.

15

Failure to mask produces the classic error of a 'shadow curve', in which a dead ear appears to have moderate hearing. • The audiogram plots frequency (Hz) on the horizontal axis, logarithmically, against hearing level (dB HL) on the vertical axis, increasing downwards — so a lower position on the chart means worse hearing.

16

Conventional symbols:

17

O for right air conduction, X for left air conduction, < and > (or [ and ]) for bone conduction, with red for the right ear and blue for the left.

18

Characteristic patterns: a high-frequency sloping loss in presbycusis, a notch at 4 kHz in noise-induced loss, a flat or rising low-frequency loss in early Ménière's disease, and Carhart's notch at 2 kHz in otosclerosis.

19

The pure-tone average of 500, 1,000 and 2,000 Hz is used to grade disability.

20

Speech Audiometry • Pure tones measure detection; speech audiometry measures the ability to hear and understand speech, which is what matters functionally and what pure-tone results cannot predict reliably. • Speech reception (recognition) threshold, SRT: the lowest level at which the patient can correctly repeat 50 % of spondee words (two-syllable words of equal stress, such as 'baseball' or 'hotdog').

21

It should agree within about 10 dB with the pure-tone average, and a large discrepancy suggests non-organic (functional) hearing loss or a technical error — the SRT's principal value being as a cross-check on the pure-tone audiogram. • Speech detection (awareness) threshold, SDT: the level at which speech is merely detected as present, usually about 8-10 dB below the SRT. • Speech discrimination (word recognition) score, WRS: the percentage of phonetically balanced monosyllabic words repeated correctly at a comfortable suprathreshold level (usually 30-40 dB SL).

22

Interpretation: a high score (90-100 %) is typical of conductive loss and of normal hearing, because merely making the sound louder restores intelligibility; a reduced score indicates sensorineural loss; and rollover — a score that falls as intensity is increased further — is characteristic of retrocochlear (VIII nerve) pathology such as acoustic neuroma.

23

Speech audiometry is also used to assess candidacy for and benefit from hearing aids and cochlear implants, and in noise (the speech-in-noise test) to reflect real-world difficulty. • Other audiological tests that complete the battery: tympanometry and acoustic reflex (middle ear pressure and compliance — type A normal, type B flat in effusion, type C negative pressure), otoacoustic emissions (cochlear outer hair cell function; the basis of newborn hearing screening), and auditory brainstem response (ABR/BERA), an evoked potential giving objective thresholds and retrocochlear information in infants and in uncooperative patients.

24

Bekesy Audiometry • Bekesy audiometry, devised by Georg von Békésy, is automatic self-recording audiometry.

25

The patient holds a button and presses it while the tone is audible and releases it when it is not; the audiometer automatically decreases the intensity while the button is pressed and increases it when released, and a pen records the level against frequency.

26

The result is a zigzag tracing whose midpoint is the threshold, obtained continuously as the frequency sweeps slowly, rather than at discrete frequencies. • Two tracings are made — one with a continuous (steady) tone and one with an interrupted (pulsed) tone — and the relationship between them defines the four Jerger types, which was the classic diagnostic application:

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Type I — the two tracings overlap: normal hearing or conductive loss.

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Type II — the continuous tracing drops slightly below the interrupted one (about 5-20 dB) in the high frequencies with narrowed excursions: cochlear loss.

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Type III — the continuous tracing falls dramatically away from the interrupted one across all frequencies: retrocochlear pathology.

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Type IV — the continuous tracing lies well below the interrupted one even at low frequencies: also suggestive of retrocochlear disease.

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A Type V, in which the continuous tracing lies above the interrupted one, is regarded as a sign of non-organic (functional) hearing loss. • Advantages: automatic and self-administered, giving a continuous frequency record, sensitive to abnormal auditory adaptation (tone decay), and useful in industrial screening.

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Limitations: it requires a cooperative, attentive patient, takes longer than conventional audiometry, and has been largely superseded for diagnosis by ABR, otoacoustic emissions and imaging — so it is now primarily of historical and examination interest, although the technique survives in automated and industrial audiometry.